The concept of a chemically inducible, reversible male sterility in plants by anther-specific expression of an N-acetylphosphinothricin (N-acetyl-PPT)-specific deacetylase is described in European Patent Application EP 531 716. The deacetylase genes from Streptomyces viridochromogenes [N-acetyl-L-phosphinothricylalanylalanine (N-acetyl-PTT) deacetylase, dea] and argE from Escherichia coli (N-acetyl-L-ornithine deacetylase) used here encode proteins having specificity for N-acetyl-L-PPT. For both genes, it was possible in the case of tapetum-specific expression in plants to show the occurrence of male-sterile flowers after treatment of individual buds with N-acetyl-L-PPT. For successful use of this system, in particular in the treatment of whole plants with N-acetyl-PPT under practically relevant conditions, it is advantageous to be able to employ deacetylases having high substrate affinity. Therefore further deacetylases having high affinity for N-acetyl-PPT were sought.
The application described here is thus based on the object of making available DNA molecules which code for deacetylases. Using these deacetylases, it is possible to produce plants having plant parts which can be destroyed specifically. The production of male- or female-sterile plants is of particular interest. The object is achieved by the provision of the embodiments described in the patent claims.
The invention relates to DNA molecules which code for deacetylases or proteins with the biological activity of a deacetylase. The enzymatic properties of these proteins are described in the examples. The invention also relates to DNA molecules which encode a biologically active subfragment or a derivative. The molecules according to the invention also include fragments, derivatives or allelic variants. Fragments are understood as meaning parts which still have the biological activity of a deacetylase.
The invention additionally relates to transgenic plant cells which have been transformed using the DNA molecules according to the invention. The transgenic plant cells can be prepared according to known techniques and are regenerated to give whole plants.
The invention relates to DNA molecules encoding a protein having the biological activity of an N-acetyl-PPT deacetylase.
The invention relates in particular to DNA molecules encoding a protein having the biological activity of an N-acetyl-PPT deacetylase selected from the group consisting of
a) DNA molecules which code for a protein having the amino acid sequence indicated under SEQ ID NO: 2 and fragments and/or derivatives thereof;
b) DNA molecules which code for a nucleotide sequence indicated under SEQ ID NO: 1 or sequences which deviate from this sequence within the bounds of code degeneracy;
c) DNA molecules which code for a protein having the amino acid sequence indicated under SEQ ID NO: 4 or fragments and/or derivatives thereof and
d) DNA molecules which code for a nucleotide sequence indicated under SEQ ID NO: 3 or sequences which deviate from this sequence within the bounds of code degeneracy.
The invention additionally relates to the microorganisms Stenotrophomonas sp. (DSM 9734) and Comamonas acidovorans (DSM 11070) identified and deposited according to the method described in this application.
The invention relates in particular to plant cells or plants which contain the DNA molecules according to the invention.
Of particular interest are processes for the production of plants with specifically destroyable parts by means of specific expression of a deacetylase gene and processes for the production of male- or female-sterile plants by means of specific expression of a deacetylase gene.
The present application thus also relates to:
(1) the specific concentration of microorganisms having high N-acetyl-PPT deacetylase activity
(2) the isolation of corresponding deacetylase genes
(3) the purification and characterization of the proteins having high N-acetyl-PPT deacetylase activity encoded by these genes
(4) the expression of the deacetylase genes in plants.
The present application additionally relates to:
the DNA molecules which code for enzymes having high N-acetyl-PPT deacetylase activity
the proteins which are encoded by these genes
the expression of these deacetylase genes in plants.
From soil samples, bacteria can be concentrated in mineral medium with chitin as the sole carbon source which are able to cleave N-acetyl-PPT with high effectiveness. In this manner, 2 bacterial strains were isolated as pure cultures: Stenotrophomonas sp. (DSM deposit No. DSM 9734) and Comamonas acidovorans (DSM deposit No. 11070).
For the conditions necessary in industrial production, however, it is substantially more advantageous to be able to employ a purified enzyme.
The present application comprises novel L-N-acetyl-PPT specific deacetylases, a novel and effective process for the purification and characterization of this enzyme from a concentration culture of soil microorganisms, and the use of this deacetylase.
The invention thus further relates to:
1. A deacetylase having
a molecular weight of 20,000 to 100,000 Daltons
a pH optimum of 6.5-10.0
a substrate specificity to L-N-acetyl-phosphinothricin.
2. A process for the preparation of a deacetylase, which comprises culturing a microorganism which does not form spores in a medium comprising crab chitin, and isolating the deacetylase from these microorganisms.
3. The use of the deacetylase characterized under 1. for the production of male-sterile plants and for the stereoselective preparation of L-phosphinothricin.
The invention relates in particular to an enzyme which has a temperature optimum which lies between 30xc2x0 C. and 50xc2x0 C.
The process according to the invention for the preparation of the deacetyl-ases is preferably carried out using microorganisms selected from the group which consists of the microorganisms described in the application.
Crab chitin can be obtained as described by Shimahara, Kenzo and Takiguchi, Yasuyuki (Methods in Enzymology, Vol 161, pages 417-423, 1988) or is commercially available from Sigma.
To purify the deacetylase, the microorganism is cultured in a nutrient medium optimal for its growth. The microorganism is cultured aerobically, for example submerse with shaking or stirring in shaker flasks or fermenters, if appropriate with introduction of air or oxygen. The fermentation can be carried out in a temperature range from approximately 20 to 40xc2x0 C., preferably at approximately 25 to 37xc2x0 C., in particular at 30 to 37xc2x0 C. Culture takes place in a pH range between 5 and 8.5, preferably between 5.5 and 8.0.
Under these conditions, the microorganisms in general show a noticeable accumulation of the enzyme after 1 to 3 days. The synthesis of the deacetylase begins with the log phase. The production of the enzyme can be monitored with the aid of activity tests by HPLC analysis or photometrically. The nutrient solution used for the production of the transaminase contains 0.2 to 5%, preferably 0.5 to 2%, of crab chitin and inorganic salts.
Inorganic salts which the nutrient medium can contain are, for example, chlorides, carbonates, sulfates or phosphates of the alkali metal or alkaline earth metals, iron, zinc and manganese, but also ammonium salts and nitrates.
According to the invention, effective amounts of the deacetylase can be employed in free or immobilized form for the deacetylation, preferably it is employed in plants which express dea genes.
For fixation, the known processes are suitable, such as the processes described in German Offenlegungsschriften 32 37 341 and 32 43 591.
The enzyme can be isolated and purified by classical methods by means of decomposition by ultrasound and French press, ammonium sulfate precipitation, ion exchangers, affinity chromatography and gel permeation.
The enzyme preparation can have a molecular weight of 20,000 to 100,000 Daltons, preferably 30,000 to 80,000, in particular 40,000 to 70,000 Daltons. The pH optimum of the enzyme product lies in the pH range 6.0 to 10.0, in particular 7.0 to 8.0. The temperature optimum of the enzyme lies between 30 and 50xc2x0 C., in particular between 35 and 45xc2x0 C.
The genes coding for the deacetylases were cloned in E. coli. In the case of the deac1 gene from Stenotrophomonas sp., a phagemid expression bank from genomic DNA in E. coli was screened for N-acetyl-PPT-specific deacetylase activity. The deac2 gene from Comamonas acidovorans was cloned with a genomic bank by complementation of an E. colixe2x80x94argE mutant.
The amino acid sequences derived from the DNA sequences of the two genes are similar to one another and additionally have homology to hippurate hydrolases, as are known from protein databanks.
The high substrate affinity of the deac1 protein for N-acetyl-L-PPT (Km=670 xcexcM) makes itself noticeable in the transgenic plants by a high sensitivity of the tissue to this substance. Thus in constitutive expression of the deac gene plants can be obtained whose leaves still react sensitively to concentrations of up to 0.4 mg/ml of N-acetyl-D,L-PPT(=0.2 mg/ml of L-enantiomer). In the case of tapetum-specific expression, the induction of male-sterile flowers was achieved by treatment of the buds with 2 mg/ml of N-acetyl-D,L-PPT(=1 mg/ml of L-enantiomer). The results described relate to greenhouse plants. On the basis of the low substance concentrations, if required, under open air conditions a higher dose of around 5-10-fold is possible without problems.